User:Tommie Hata/Introduction to Protein Engineering-Subtilisin

This Protein Engineering module has been developed using material from Dr. Scott Banta's course, Protein Engineering (chemical engineering department at Columbia University).



Introduction
The structure to the right is subtilisin BPN' and streptomyces subtilisin inhibitor (PDB ID: 2sic). The subtilisin enzyme is colored white while the Streptomyces subtilisin inhibitor is colored yellow. By minimizing the inhibitor to a fragment that is bound in the subtilisin active site, we can take a closer look at the active site. Enzyme-inhibitor structures are common in the Protein Data Bank (PDB)compared to enzyme-substrate structures. This is because enzyme-substrate complexes are often transient. The inability to form a stable enzyme-substrate complex makes it difficult to grow a crystal for structural determination through X-ray crystallography. In comparison, enzyme inhibitors bind their targets with much higher affinity (low dissociation constant) which makes the enzyme-inhibitor complex more favorable to crystallize for structure determination.

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Function
Subtilisin is a serine protease. The catalytic triad in the active site is made up of aspartic acid (Asp32), histidine (His64), and serine (Ser221). Subtilisins are the proteases used in many laundry and dishwashing detergents. In 2002 alone, 900 tons of subtilisin was produced and used in the EU. Subtilisin received the first US patent for an engineered protein in 1988.

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Engineered features
Subtilisins already possess a very high natural stability, partially because they are extracelluar enzymes optimized to work outside of the cell. Yet there are additional characteristics that have been engineered into industrial subtilisins for turn them into better products.

A calcium-independent thermostable subtilisin BPN mutant (PDB ID: 1gnv)

 

, which has been minimized in the view to highlight the chain bound in the active site of subtilisin.

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Reference
Structures

Features and industrial use

Engineering